The long term objective of the proposed work is the elucidation of the mechanism of DNA replication in animal mitochondria. This may be accomplished when purified enzymes are reconstituted in vitro to yield replication complexes capable of initiation and elongation of DNA synthesis. The studies will provide the framework for research to determine the regulation of these processes as they occur in vivo. The current proposal focuses on the major replicative enzyme in mitochondria, DNA polymerase gamma. A combined approach involving biochemical, immunological and molecular biological methods will be employed to undertake mechanistic and structure-function analysis of the mitochonchial DNA polymerase and to done and overproduce its two subunits. Secondary goals involve isolation of a mitochondrial RNA polymerase / DNA primase and examination of the co-ordination of RNA priming and DNA synthesis on mitochondrial DNA templates. The control of animal cell reproduction during normal development, and the loss of control during cancerous development, is of central importance in the processes of human growth, aging, and disease. Prior to every cell division, the DNA complement of each cell is duplicated. Because DNA replication and cell division are tightly coupled, an understanding of the mechanisms of DNA replication functioning in animal cells is essential to our understanding of these basic processes. Mitochondrial biogenesis proceeds in parallel with cell proliferation, but it is neither tightly coupled to mitochondrial DNA replication nor to the cell cycle. Nevertheless, because both the DNA content of the mitochondrion and the number of mitochondria in cells remain relatively constant, specific regulatory mechanisms are likely required to couple mitochondrial DNA replication and biogenesis to nuclear DNA replication and cell division. A detailed analysis of the key enzyme involved in mitochondrial DNA replication, gamma polymerase, will represent a major contribution toward an eventual understanding of mitochondrial biogenesis.